Nonintegrating lentiviral viral vectors (NILVs) present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling the short-term expression of potentially hazardous gene products. In recent studies, NILVs proved efficient at inducing an antigen specific immune response in vivo and are currently being used as a platform to deliver zinc finger nucleases to mediate site-specific gene editing. However, additional improvements in the NILV system are required to render this promising gene delivery system suitable for human clinical trials. These improvements include: a) increasing episomal gene expression, b) reducing illegitimate vector integration, c) developing an efficient and nonimmunogenic gene regulation system, and d) establishing an efficient vector production system. To achieve these improvements, we outline here a research proposal consisting of four aims. In aim 1 we propose to develop a novel NILV deleted of the cis inhibitory elements recognized by the host LSF-YY1 and AP-4 transcriptional repressor complexes. This proposed modification will alleviate the transcriptional silencing typical of NILVs and will improve their efficacy. Aim 2 will focus on the development of a novel inducible NILV system premised on alternative splicing. In contrast to currently used inducible systems, the novel splicing-regulated NILVs do not contain a synthetic transactivator, which could potentially induce a cell-mediated immune response. In aim 3, we will focus on characterization and in vivo testing of a novel polypurine tract (PPT) deleted NILV, which exhibits reduced illegitimate integration. In the last aim, we will establish the first integrase-deficient stable packaging cell line, which will produce high-titer NILVs bearing the improvements described in aims 1-3. All new vectors containing the different modifications will carry an improved human factor IX (hFIX) cDNA. The vectors will be produced either by transient transfection or by the novel stable packaging cell line, and their ability to cure FIX deficiency will be tested in a hemophilia B mouse model.